Long Interspersed Element-1 (LINE-1 or L1) is an abundant non-Long Terminal Repeat (non-LTR) I retrotransposon that comprises about 17% of human DNA. The average human genome contains -80-100 retrotransposition-competent Lls (RC-Lls) and these elements encode enzymatic activities (e.g., reversel transcriptase and endonuclease) required for their mobility (i.e., retrotransposition). L1 retrotransposition can be mutagenic, as deleterious insertions in both germ line and somatic tissues have caused disease. The RC-L1 encoded proteins also occasionally can function in trans to mobilize either non-autonomous retrotransposons (e.g., Alu and SVA elements) or cellular mRNAs, resulting in processed pseudogene formation. Deleterious Alu and SVA insertions also are implicated in human disease. Thus, either directly or by the promiscuous mobilization of cellular RNAs, RC-Lls are potent mutagens. Despite the mutagenic potential of RC-Lls, relatively little is known about their retrotransposition mechanism. My lab has developed a high-throughput assay as well as molecular biological and biochemical approaches to monitor L1 retrotransposition in cultured mammalian cells. Here, we will use these approaches to elucidate the molecular mechanism of L1 retrotransposition. The Specific Aims of this proposal are 1) To identify and characterize sequences in the L1 5' UTR, L1 RNA, and the Ll-encoded proteins, which are critical for retrotransposition; 2) To determine the molecular mechanism of Ll-mediated trans-complementation; and 3) To characterize L1 ribonucleoprotein particles. The long-term goal of this project is to gain a fundamental understanding of how L1 retrotransportation contributes to human disease and genetic diversity.